Phospholipase C-η2 interacts with nuclear and cytoplasmic LIMK-1 during retinoic acid-stimulated neurite growth

Abstract

Neurite growth is central to the formation and differentiation of functional neurons, and recently, an essential role for phospholipase C-η2 (PLCη2) in neuritogenesis was revealed. Here we investigate the function of PLCη2 in neuritogenesis using Neuro2A cells, which upon stimulation with retinoic acid differentiate and form neurites. We first investigated the role of the PLCη2 calcium-binding EF-hand domain, a domain that is known to be required for PLCη2 activation. To do this, we quantified neurite outgrowth in Neuro2A cells, stably overexpressing wild-type PLCη2 and D256A (EF-hand) and H460Q (active site) PLCη2 mutants. Retinoic acid-induced neuritogenesis was highly dependent on PLCη2 activity, with the H460Q mutant exhibiting a strong dominant-negative effect. Expression of the D256A mutant had little effect on neurite growth relative to the control, suggesting that calcium-directed activation of PLCη2 is not essential to this process. We next investigated which cellular compartments contain endogenous PLCη2 by comparing immunoelectron microscopy signals over control and knockdown cell lines. When signals were analyzed to reveal specific labeling for PLCη2, it was found to be localized predominantly over the nucleus and cytosol. Furthermore in these compartments (and also in growing neurites), a proximity ligand assay revealed that PLCη2 specifically interacts with LIMK-1 in Neuro2A cells. Taken together, these data emphasize the importance of the PLCη2 EF-hand domain and articulation of PLCη2 with LIMK-1 in regulating neuritogenesis.

Phospholipase C families: Common themes and versatility in physiology and pathology

“…Using the NeuroA cells as a model for neuronal differentiation by retinoic acid, it was found that PLCε2 activity was required for differentiation. A mutant devoid of PLC activity inhibited differentiation as did knockdown by siRNA [387]. LIMK-1 (LIM domain kinase 1) was found to interact with PLCε2 [387,389].…”

“…Because PI(4,5)P 2 has multiple functions including regulation of ion channels and actin cytoskeleton and is required for exocytosis, PLCε2 could be an important modulator of neuronal function. The function of PLCε2 has been explored in neuronal cultured cell-lines and results suggest that it may be involved in neuronal differentiation and vesicle exocytosis in neuroendocrine cells [387,388]. Using the NeuroA cells as a model for neuronal differentiation by retinoic acid, it was found that PLCε2 activity was required for differentiation.…”

The impact of oxytocin on neurite outgrowth and synaptic proteins in Magel2‐deficient mice

“…In this study, we observed decreased expression of Limk1 and Tiam1 , which are enzymes known to be associated with rearranging of the cytoskeleton (see scheme in Figure 11) and interact with the retinoid receptor function (Ishaq et al., 2011). Activation of LIMK1 is associated with retinoic acid‐stimulated neurite outgrowth (Arastoo et al., 2016). LIMK1 regulates actin dynamics by phosphorylating cofilin, which contributes to processes of neurite elongation (Reichova et al., 2018).…”

Abnormalities in interactions of Rho GTPases with scaffolding proteins contribute to neurodevelopmental disorders

“…Phosphorylation of LIM kinases integrates signals from a number of Rho GTPases and serves as a link to differentiation processes. One recent study has found that stimulation of Neuro2A cells with retinoic acid results in the differentiation and formation of neurites with corresponding changes of LIM kinase interaction capabilities (Arastoo et al, ). Another downstream effector of Rho GTPases represented by p21‐activated kinases (PAKs), which convey signals to the direct regulators of the actin cytoskeleton (Woolfrey & Srivastava, ).…”

“…It is widely discussed that inhibition of RhoA/ROCK in the central nervous system promotes axon growth and regeneration (Fujita & Yamashita, 2014). Some studies claim that ROCK inhibition may be a promising target for neuroprotection and axon regeneration (Van de One recent study has found that stimulation of Neuro2A cells with retinoic acid results in the differentiation and formation of neurites with corresponding changes of LIM kinase interaction capabilities (Arastoo et al, 2016). Another downstream effector of Rho GTPases represented by p21-activated kinases (PAKs), which convey signals to the direct regulators of the actin cytoskeleton (Woolfrey & Srivastava, 2016).…”